The human Caliciviruses Norovirus and Sapovirus are leading causes of acute, non-bacterial gastroenteritis. In contrast to Norovirus, Sapovirus is known to give infections mainly in infants and young children, however Sapovirus is increasingly found in the adult populations as well (Johansson et al., 2005, A nosocomial sapovirus-associated outbreak of gastroenteritis in adults. Scand J Infect Dis. 37(3):200-4). Noroviruses are non-cultivatable human Caliciviruses that have emerged as the single most important cause of epidemic outbreaks of nonbacterial gastroenteritis (Hardy, 1999, Clin Lab Med. 19(3):675-90). The clinical significance of Noroviruses was under-appreciated prior to the development of sensitive molecular diagnostic assays. The cloning of the prototype genogroup I Norwalk virus (NV) genome and the production of virus-like particles (VLPs) from a recombinant Baculovirus expression system led to the development of assays that revealed widespread Norovirus infections (Jiang et al. Norwalk Virus Genome Cloning and Characterization. Science 1990; 250: 1580-1583; Jiang et al. 1992, J. Virol. 66(11):6527-32).
Noroviruses and Sapoviruses are single-stranded, positive sense RNA viruses that contain a non-segmented RNA genome. The viral genome encodes three open reading frames, of which the latter two specify the production of the major capsid protein and a minor structural protein, respectively (Glass et al., The Epidemiology of Enteric Caliciviruses from Human: A Reassessment Using New Diagnostics. J Infect Dis 2000; 181 (Sup 2): S254-S261). When expressed at high levels in eukaryotic expression systems, the capsid protein of NV, and certain other Noroviruses and Sapoviruses, self-assembles into VLPs that structurally mimic native Norovirus virions. When viewed by transmission electron microscopy, the VLPs are morphologically indistinguishable from infectious virions isolated from human stool samples.
Although Norovirus and Sapovirus cannot be cultivated in vitro, due to the availability of VLPs and their ability to be produced in large quantities, considerable progress has been made in defining the antigenic and structural topography of the Norovirus capsid. VLPs preserve the authentic conformation of the viral capsid protein while lacking the infectious genetic material. Consequently, VLPs mimic the functional interactions of the virus with cellular receptors, thereby eliciting a strong host immune response while lacking the ability to reproduce or cause infection. In conjunction with the NIH, Baylor College of Medicine studied the humoral, mucosal and cellular immune responses to Norovirus VLPs in human volunteers in an academic, investigator-sponsored Phase I clinical trial. Orally administered VLPs were safe and immunogenic in healthy adults (Ball et al. 1999; Tacket et al. 2003). At other academic centers, preclinical experiments in animal models have demonstrated enhancement of immune responses to VLPs when administered intranasally with bacterial exotoxin adjuvants (Guerrero et al. 2001, Recombinant Norwalk Virus-like Particles Administered Intranasally to Mice Induce Systemic and Mucosal (Fecal and Vaginal) Immune Responses. J Virol 2001; 75: 9713; Nicollier-Jamot et al. 2004, Recombinant Virus-like Particles of a Norovirus (Genogroup II Strain) Administered Intranasally and Orally with Mucosal Adjuvants LT and LT(R192G) in BALB/c Mice Induce Specific Humoral and Cellular Th1/Th2-like Immune Responses. Vaccine 2004; 22:1079-1086; Periwal et al. 2003, Enhances Systemic and Mucosal Immune Responses to Recombinant Norwalk Virus-like Particle Vaccine. Vaccine 2003; 21: 376-385).
Small-scale methods for purifying Norovirus VLPs have been described in the literature. For example, Norwalk virus VLP purification by ultracentrifugation has been described (Jiang et al. 1990; 1992) and is commonly employed by the Norovirus investigators in the field. However, while VLPs purified by ultracentrifugation have been used in human clinical trials, the method is not suitable for producing commercial scale quantities of Calicivirus VLPs. Consequently, there remains a need to provide a scalable and efficient purification system capable of purifying VLPs from various biological sources.